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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 閔明源 | zh_TW |
| dc.contributor.advisor | Ming-Yuan Min | en |
| dc.contributor.author | 鄭淵仁 | zh_TW |
| dc.contributor.author | Yuan Ren Cheng | en |
| dc.date.accessioned | 2024-03-07T16:13:34Z | - |
| dc.date.available | 2024-03-08 | - |
| dc.date.copyright | 2024-03-07 | - |
| dc.date.issued | 2024 | - |
| dc.date.submitted | 2024-02-17 | - |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/92133 | - |
| dc.description.abstract | 背景： 受Martin Chalfie 對觸覺接受器神經元（TRNs）和機械活化通道研究的啟發，本研究聚焦於酸敏感離子通道（ASICs），特別是ASIC3，在背根神經節（DRG）內表達Parvalbumin的本體感受神經元中的機械轉導作用。有別於Piezo channel 在背根神經節中的直接接觸模式，本研究將「牽引力模型」來研究神經元機械感應。
方法： 本研究採用先進的機械力控制膜片鉗電生理技術、條件性基因敲除實驗，深入研究ASIC3在本體感知中的功能。引入使用聚二甲基矽氧烷（PDMS）覆蓋玻片的新方法模擬生理彈性，以準確研究這些特定DRG神經元的機械敏感性。 結果： 本研究結果中顯示了ASIC3在酸性條件下在機械轉導中的重要作用。在表達Parvalbumin的本體感受神經元中，ASIC3的基因敲除導致本體感知受損，以及因神經突伸長牽引的機械轉導受到破壞。同時使用ASIC3專一性抑制劑APETx2的應用凸顯了ASIC3通道的功能細節。APETx2的處理改變了這些神經元的機械敏感性，強調了ASIC3在牽引力模型機械感應中的關鍵作用，以及受到環境音子影響的調控能力，並指出ASIC家族內的多樣性功能。 結論： 本研究確立了ASIC3作為本體感受神經元中機械轉導的核心元素。APETx2的研究不僅強調了ASIC3在本體感知中的關鍵作用，還揭示了ASIC家族的異質性和廣泛的功能範疇。這種異質性表明ASIC通道在各種生理和病理條件下的複雜相互作用，突顯了它們作為感覺處理障礙治療的潛力。，考慮到ASIC通道多樣且細膩的角色這些發現為針對性介入機械感應轉導打開了新的視角。 關鍵字：酸敏感離子通道第三型、機械力傳導、本體感覺 | zh_TW |
| dc.description.abstract | Background: Inspiring by Martin Chalfie’s study of touch receptor neurons (TRNs) and mechanically activated ion channels, this research focuses on the role of Acid-Sensing Ion Channels (ASICs), especially ASIC3, in mechanotransduction within parvalbumin-expressing proprioceptive neurons in the dorsal root ganglia (DRG). The study contrasts the ''tether model'' of mechanosensation with the bilayer model represented by Piezo channels.
Methods: Employing advanced electrophysiological techniques, genetic axonal tracing, and conditional knockout experiments, the research scrutinizes ASIC3''s function in proprioception. Introducing a novel method using polydimethylsiloxane (PDMS) coverslips simulates physiological elasticity, facilitating an accurate study of the mechanosensitivi6ty of these specific DRG neurons. Results: The study reveals ASIC3''s substantial role in mechanotransduction under acidotic conditions. In parvalbumin-expressing proprioceptors, ASIC3 knockout results in impaired proprioception and disrupted neurite stretch-induced mechanotransduction. The application of APETx2, an ASIC3 inhibitor, highlighted the functional nuances of ASIC3 channels. Treatment with APETx2 modified the mechanosensitivity of these neurons, emphasizing ASIC3''s critical role in the tether model of mechanosensation, regulation of acidosis in the microenvironment, and indicating the diverse functionality within the ASIC family. Conclusion: This research establishes ASIC3 as a central element in mechanotransduction within proprioceptive neurons. The APETx2 study not only underscores ASIC3''s pivotal role in proprioception but also reveals the ASIC family''s heterogeneity and broad functional spectrum. This heterogeneity suggests a complex interplay of ASIC channels in various physiological and pathophysiological conditions, highlighting their potential as therapeutic targets for sensory processing disorders. The findings open new perspectives for targeted interventions in mechanosensory transduction, considering ASIC channels'' diverse and nuanced roles. Keywords: ASIC3, mechanotransduction, proprioception | en |
| dc.description.provenance | Submitted by admin ntu (admin@lib.ntu.edu.tw) on 2024-03-07T16:13:34Z No. of bitstreams: 0 | en |
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| dc.description.tableofcontents | 口試委員審定書 i
致謝 ii 中文摘要 iii Abstract iv Abbreviation vi Chapter 1. General Introduction 1 Preface : The basis of tethered model mechanotransduction 2 1. Molecular Mechanisms Involved in Neurosensory Mechanotransduction 3 1.1 Discovery of Piezo Channels and Deficiency of Piezo Gene Knockout 3 1.2 Acid-Sensing Ion Channels (ASICs) and their relative physiological function 5 1.2.1 ASICs member and their expression patterns in mice 5 1.2.2 Phenotyping ASIC-Knockout Mice 8 2. Controversial results and limitation of current methods 11 2.1 Unexpected mechanotransduction phenotype in ASIC subtype knockout mice 11 2.2 Effect of amiloride and its analogues in stretch evoke afferent discharge 12 2.3 The methods demonstrate Piezo 2 as a mechanotransducer channel 13 2.4 Discrepancy of previous study indicates the limitation of current methods 15 2.5 Proprioceptive DRG neurons could be an ideal cellular model 17 2.6 Potential roles of ASIC3 in proprioceptive neurosensory mechanotransduction 18 3. Central Hypothesis 19 Chapter 2. Materials and Methods 22 1. Preparation of elastomeric substrate 23 1.1. PDMS Preparation (3 days before DRG culture) 23 1.2. Drying and Storage 23 1.3. Sterilization 23 1.4. Ethanol Sterilization and Washing 24 1.5. Fibronectin Coating (2 hours, one day before cell culture) 24 1.6. Washing of PDMS Coverslips (1 hour, one day before cell culture) 24 2. Preparation of dorsal root ganglion primary culture 24 2.1. Preparation and Harvesting (20–30 minutes) 24 2.2. Primary Culture (~5 hours) 25 2.3. Trituration and Seeding 25 2.4. Incubation and Neurite Growth 25 3. Whole-cell Mechano-Patch Clamp equipment settings 26 3.1 Recording Chamber 26 3.2 Perfusion System 26 3.3 Recording Electrode 27 3.4 Indentation electrode 27 3.5 Grounding for Noise Reduction 28 3.6 PDMS Coated Coverslip and Neurite-Bearing Primary Culture 28 3.7 Whole-cell Mechano-Patch Clamp Process 28 3.7.1 Preparation of Recording Electrode 29 3.7.2 Bath Perfusion 29 3.7.3 Mechanical Stretching Setup 29 3.7.4 Recording Chamber Setup 29 3.7.5 Microscope Visualization 29 3.7.6 Whole-Cell Recording 30 3.7.7 Action Potential Recording 30 3.7.8 Indentation Procedure 30 3.7.9 Multiple Indentations 30 3.2.1 Animal Procedures: 31 3.2.2 Generation of Asic3-Knockout/eGFP-f-Knockin Mice: 31 4. Generation of Asic3f/f Mice 32 5. Immunostaining Methodology 32 6. Single-cell RT–PCR Procedure 34 7. Electrophysiology and Whole-cell Patch-clamp Recordings 35 8. Rheobase Analysis of AP Threshold 36 9. Mechanically Activated Current Recording 36 9.1 Mechanically Activated Currents during Acidosis 37 9.2 ASIC3 Dependency with Pharmacological Testing 37 10. Grid-Walking Task 37 11. Balance Beam Walking Task 38 12. Statistical Analysis 38 Chapter 3. Results 40 1 Develop a software to control the indentation process and motorize micro-manipulator 41 1.1 Serial Port Communication 41 1.2 Command Handling 41 1.3 Operational Logic 41 1.4 Main Application Flow 42 2. Precise timing control and consistence of substrate deformation neurite stretch (SDNS) 42 2.1 Potential failure mode of mechanical stimulus 43 2.2 Repeatable localized mechanical stimulation 43 2.3 Membrane potential changes evokes SDNS 45 2.4 Mechanical activated action potential threshold of SDNS 45 2.5 Summary of SDNS control 46 3. Heterogeneous Expression of ASIC3 in DRG and its involvement in proprioception 47 3.1 Widespread ASIC3 Expression in Parvalbumin-Positive DRG Neurons 48 3.2 Characterizing Acid-Sensing Ion Channel 3 (ASIC3) Knockout in Pv+ DRG Neurons 49 3.3 Tether Mode Sensing Role of ASIC3 in Proprioceptive DRG Neurons 51 3.4 ASIC3 KO Impairs Grid and Balance Beam Walking: 54 3.5 Brief summary of ASIC3 in proprioception 56 4. Probing the Effect of Acidosis on ASIC3 related Tether-Mode Mechanotransduction 57 4.1 Detailed Analysis of Pv+ DRG Neurons in MSs and GTOs 57 4.2 Impact of Acidic Environment on Mechanically Activated Currents by SDNS 58 4.3 ASIC3's Influence in Tether-Mode Mechanotransduction under Acidic Conditions 59 4.4 Summary of ASIC3 in Acidic Modulation of Proprioceptive Mechanotransduction 60 Chapter 4. Discussion 62 1. Molecular Basis of Chemo-Sensing and Mechano-Sensing in ASICs: 63 1.1 Chemo-Sensing in ASICs: 63 1.2 Non-acid Chemo-Sensing in ASICs 65 1.3 Lactate Sensing in ASICs 65 1.4 Mechano-Sensing in ASICs 66 1.5 ASICs as tunable somato-sensory transducers 67 2 The Mechano-Sensing Apparatus: Tether Mode Neurosensory Mechanostransduction 69 2.1 Distribution of ASIC3 in mechanosensory organs and DRG neurons 69 2.3 The conditional knockout ASIC3 in proprioceptive neurons results in dysfunction in neurosensory mechanotransduction 70 2.4 Tentative role of ASIC3 in tether mode mechanotransduction 71 3 Unraveling the Impact of Acidosis and Environmental Factors on Tether Mode Mechanotransduction: Insights from Proprioceptive Neurons 73 3.1 Evidence of ASIC3 Involvement in Alternative Environmental Factors: Acidosis in Proprioceptive Neurosensory Mechanotransduction 73 3.2 Tentative mechanisms of Acidosis-Induced Structural Changes in ASIC3 involved heteromeric channels and their impact on proprioceptive mechanosensory function 75 Chapter 5. Conclusion 78 Chapter 6. Reference 80 Chapter 7. Figures 98 Figure 1 PDMS with surface modification 100 Figure 2 Main Application Flow Control: Central hub for managing operational processes 102 Figure 3 Software Control Logic Overview 104 Figure 4 Analysis of Indentation Failure Scenarios 106 Figure 5 Monitoring of Localized Indentation-Induced Mechanical Disturbances in PDMS Using Fluorescent Beads 108 Figure 6 Analysis of GFP Expression in ASIC3-Expressing DRG Neurons of Asic3-KO/eGFP-f-KI (Asic3▵EGFPf/EGFPf) Mice 110 Figure 7 Distribution of ASIC3-Expressing DRG Neurons in Muscular and Proprioceptive Systems 112 Figure 8. PvCre-CAG reporter indicating parvalbumin expression DRG neurons and it’s distinct with Nav1.8 Cre-CAG nociceptive neurons. 114 Figure 9 Impact of Asic3 Conditional Knockout in DRG Pv+ Neurons 116 Figure 10. The presence of ASIC and Piezo gene expressions in mouse DRG neurons. 118 Figure 11 Role of ASIC3 in Neurite Stretch-Induced Firing in Pv+ DRG Neurons 119 Figure 12 Neurite Stretch Responses in Wildtype and Asic3-Null Pv+ DRG Neurons 122 Figure 13 Impact of ASIC3 Blockade on SDNS-Induced Action Potentials in Wild-Type Pv+ DRG Neurons 124 Figure 14 Comparative Rheobase Analysis of Action Potential Threshold in Wildtype and Asic3-Null Pv+ DRG Neurons 126 Figure 15 Behavioral Assessment of Proprioception in Mice with Asic3 Deficiency 128 Figure 16 Identification of Parvalbumin+ Proprioceptive Muscle Afferents with Neurofilament Heavy Chain Immunoreactivity and Specialized Mechanosensing Structures 130 Figure 17 Overview of the SDNS Technique with acidosis perfusion 132 Figure 18. Mild Acidosis Effect on SDNS Currents: An Experimental Analysis 134 Figure 19. Acidic Perfusion's effect on SDNS Currents in Response to Force 136 Figure 20. pH-Dependent Modulation of SDNS-Induced Currents in Pv+ DRG Proprioceptors Under Mild Acidosis 139 Figure 21. Role of ASIC3 in SDNS-Induced Currents Across Proprioceptor Subtypes" 142 Figure 22. Influence of APETX2 and Mild Acidosis (pH 6.8) on SDNS-Induced Currents in Distinct Pv+ DRG Neuron Subtypes 145 Figure 23. Classification of Pv+ DRG Neurons Based on SDNS-Induced and Acid-Induced Currents 148 Figure 24. Theoretical Models on ASIC3's Role in Mild Acidosis-Induced Attenuation of SDNS Currents 151 Table 1. Sequence of single cell RT-PCR nested primers 152 Table 2. AP Profile of ISDNS recording Parvalbumin expression DRG neurons 153 APPENDIX 1. Acid-sensing ion channels: dual function proteins for chemo-sensing and mechano- sensing 155 Appendix 2. Probing localized neural mechanotransduction through surface-modified elastomeric matrices and electrophysiology 170 Appendix 3. Evidence for the involvement of ASIC3 in sensory mechanotransduction in proprioceptors 180 Appendix 4. Probing the Effect of Acidosis on Tether-Mode Mechanotransduction of Proprioceptors 196 | - |
| dc.language.iso | en | - |
| dc.title | 探討組織酸化對本體感覺神經元中牽引力學傳導的影響 | zh_TW |
| dc.title | Probing the Effect of Acidosis on Tether-Mode Mechanotransduction of Proprioceptors | en |
| dc.type | Thesis | - |
| dc.date.schoolyear | 112-1 | - |
| dc.description.degree | 博士 | - |
| dc.contributor.coadvisor | 陳志成 | zh_TW |
| dc.contributor.coadvisor | Chih-Cheng Chen | en |
| dc.contributor.oralexamcommittee | 潘建源;湯志永;徐經倫;林以文 | zh_TW |
| dc.contributor.oralexamcommittee | Chien-Yuan Pan;Chih-Yung Tang;Ching-Lung Hsu;Yi-Wen Lin | en |
| dc.subject.keyword | 酸敏感離子通道第三型,機械力傳導,本體感覺, | zh_TW |
| dc.subject.keyword | ASIC3,mechanotransduction,proprioception, | en |
| dc.relation.page | 218 | - |
| dc.identifier.doi | 10.6342/NTU202400632 | - |
| dc.rights.note | 同意授權(全球公開) | - |
| dc.date.accepted | 2024-02-17 | - |
| dc.contributor.author-college | 生命科學院 | - |
| dc.contributor.author-dept | 生命科學系 | - |
| 顯示於系所單位： | 生命科學系 | |
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